US6143566A - Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof - Google Patents
Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof Download PDFInfo
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
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- C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
Definitions
- This invention relates generally to methods of modifying genes with specificity in recombination deficient cells by transiently enabling homologous recombination in the cells. Included in the invention are conditional replication shuttle vectors which bestow transient recombination capabilities to an otherwise recombination deficient cell.
- the independent origin based cloning vectors containing the modified genes and methods of using the independent origin based cloning vectors containing the modified genes are also included in the present invention.
- the size of the genomic DNA that can be readily manipulated in vitro and introduced into the germline can be a critical determinant of the outcome of the functional analysis of a gene since elements that are important for high level, tissue specific and position-independent expression of the transgene may be located at a long distance from the gene itself [Dillon et al., Trends Genet. 9:134 (1993); Kennison, Trends Genet. 9:75 (1993); Wilson et al., Annu. Rev. Cell. Biol. 6:679 (1990)].
- the use of such large genomic transgenes has several practical problems.
- the size of the transgene is presently limited due to constraints on the sequence length that can be cloned and stably maintained in a conventional plasmid or a cosmid.
- DNA sequences suspected of being nonessential are often omitted when designing the constructs to be transferred because of the size limitation.
- in vitro manipulations of large DNAs oftentimes lead to mechanical shear [Peterson et al., TIG 13:61-66].
- Yeast artificial chromosomes allow large genomic DNA to be modified and used for generating transgenic animals [Burke et al., Science 236:806; Peterson et al., Trends Genet. 13:61 (1997); Choi, et al., Nat. Genet., 4:117-223 (1993), Davies, et al., Biotechnology 11:911-914 (1993), Matsuura, et al., Hum. Mol. Genet., 5:451-459 (1996), Peterson et al., Proc. Natl. Acad. Sci., 93:6605-6609 (1996); and Schedl, et al., Cell, 86:71-82 (1996)].
- YACs Yeast artificial chromosomes
- YACs have certain advantages over these alternative large capacity cloning vectors [Burke et al., Science, 236:806-812 (1987)].
- the maximum insert size is 35-30 kb for cosmids, and 100 kb for bacteriophage P1, both of which are much smaller than the maximal insert for a YAC.
- YACs E. coli based cloning systems based on the E. coli fertility factor that have been developed to construct large genomic DNA insert libraries. They are bacterial artificial chromosomes (BACs) and P-1 derived artificial chromosomes (PACs) [Mejia et al., Genome Res. 7:179-186 (1997); Shizuya et al., Proc. Natl. Acad. Sci. 89:8794-8797 (1992); Sicilnou et al., Nat. Genet., 6:84-89 (1994); Hosoda et al., Nucleic Acids Res. 18:3863 (1990)]. BACs are based on the E.
- coli fertility plasmid F factor
- PACs are based on the bacteriophage P1.
- the size of DNA fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules has been expanded by the advent of PACs and BACs.
- These vectors propagate at a very low copy number (1-2 per cell) enabling genomic inserts up to 300 kb in size to be stably maintained in recombination deficient hosts (most clones in human genomic libraries fall within the 100-200 kb size range).
- the host cell is required to be recombination deficient to ensure that non-specific and potentially deleterious recombination events are kept to a very minimum.
- Functional characterization of a gene of interest contained by a PAC or BAC clone generally entails transferring the DNA into a eukaryotic cell for transient or long-term expression.
- a transfection reporter gene e.g., a gene encoding lacZ, together with a selectable marker, e.g., neo, can be inserted into a BAC [Mejia et al., Genoine Res. 7:179-186 (1997).
- Transfected cells can be then detected by staining for X-Gal to verify DNA uptake.
- Stably transformed cells are selected for by the antibiotic G418.
- a cloning vector that has the capacity to contain greater than 100 kilobases of DNA, which can be readily manipulated and isolated, but still can be stably stored in libraries relatively free of rearranged clones.
- methodology for generating such cloning vectors There is also a need to apply such vectors to improve current technologies such as gene targeting.
- Gene targeting has been used in various systems, from yeast to mice, to make site specific mutations in the genome. Gene targeting is not only useful for studying function of proteins in vivo, but it is also useful for creating animal models for human diseases, and in gene therapy.
- the technique involves the homologous recombination between DNA introduced into a cell and the endogenous chromosomal DNA of the cell. However, in the vertebrate system, the rate of homologous recombination is very low, as compared to random integration.
- the only cell line that allows a relatively high homologous recombination rate and maintains the ability to populate the germline is the murine 129 embryonic stem cells (ES cells).
- mice can be generated with a targeted mutation (Gene Targeting, a practical approach Ed. by A. Joyner, IRL Press: Oxford, New York, Tokyo).
- Gene Targeting a practical approach Ed. by A. Joyner, IRL Press: Oxford, New York, Tokyo.
- the rate of homologous recombination for some gene loci in ES cells is still extremely low ( ⁇ 1%), the procedure is labor intensive, and the cost of generating targeted mutant mice is very expensive.
- gene targeting in a germline is still not possible for other vertebrates.
- the major limitation for gene targeting in vertebrate cells remain to be the low targeting frequency.
- One critical factor affecting the targeting frequency is the total length of homology.
- Deng and Capecchi (MCB, 12:3365-3371) have shown that gene targeting frequency is linearly-dependent on the logarithm of the total homology length over homology lengths of 2.8 kb to 14.6 kb. Since the curve did not plateau at the 14.6 kb homology, it is likely that incorporating greater homology lengths into the targeting vector will further increase the homologous recombination rate.
- gene targeting in TB has a very low rate, mainly due to the predominance of random integration over homologous recombination. It has been demonstrated that using a 40-50 kb linear targeting construct, a 6% targeting frequency could be obtained, whereas no targeting event was obtained at all with a smaller ( ⁇ 10 kb) targeting construct [Balasubramanian et al., J. of Bacteriology 178:273-279 (1996)]. Therefore, there is a need to construct large gene targeting constructs to allow efficient gene targeting in many biological systems.
- the present invention provides a novel and efficient method of modifying independent origin based cloning vectors for in vitro and in vivo gene expression.
- the present invention provides a method of selectively performing homologous recombination on a particular nucleotide sequence contained in a recombination deficient host cell, i.e., a cell that cannot independently support homologous recombination.
- the method employs a recombination cassette which contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination deficient host cell is induced to support homologous recombination.
- the method comprises introducing the recombination cassette into the recombination deficient host cell, and inducing the recombinantly deficient host cell to transiently support homologous recombination, thereby allowing the nucleic acid to integrate into the particular nucleotide sequence.
- unselected nucleotide sequence rearrangements and deletions which are characteristic of host cells that support homologous recombination, are not evident with restriction endonuclease digestion map analysis with a restriction enzyme such as HindIII, EcoRI, XhoI, or AvrII.
- unselected nucleotide sequence rearrangements and deletions are not evident with restriction endonuclease digestion map analysis with two or more restriction enzymes.
- the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA - .
- inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of a RecA-like protein in the host cell.
- inducing the transient expression of the RecA-like protein can be performed with a conditional replication shuttle vector.
- the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
- inducing the transient expression of the RecA-like protein comprises transforming the host cell with the TSSV at a permissive temperature, and growing the host cell at a non-permissive temperature.
- the TSSV encodes a RecA-like protein that is expressed in the host cell and supports the homologous recombination between a nucleic acid contained in a recombination cassette and the particular nucleotide sequence contained in the host cell.
- the TSSV encoding the RecA-like protein is diluted out when the host cell is grown at the non-permissive temperature.
- the permissive temperature is 30° C. and the non-permissive temperature is 43° C.
- the particular nucleotide sequence which has been selected to undergo homologous recombination is contained in an independent origin based cloning vector (IOBCV) that is comprised by the host cell, and neither the independent origin based cloning vector alone, nor the independent origin based cloning vector in combination with the host cell, can independently support homologous recombination.
- IBCV independent origin based cloning vector
- both the independent origin based cloning vector and the host cell are RecA - , and inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of the RecA-like protein to support homologous recombination in the host cell.
- the independent origin based cloning vector is a Bacterial or Bacteriophage-Derived Artificial Chromosome (BBPAC) and the host cell is a host bacterium.
- BBPAC Bacteriophage-Derived Artificial Chromosome
- inducing the transient expression of the RecA-like protein is performed with a conditional replication shuttle vector that encodes the RecA-like protein.
- the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
- TSSV temperature sensitive shuttle vector
- the permissive temperature is 30° C. and the non-permissive temperature is 43° C.
- the RecA-like protein is controlled by an inducible promoter and the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in the host cell.
- the RecA-like protein is controlled by a constitutive promoter with the transient expression induced by the TSSV.
- the TSSV also comprises a recombination cassette and a first gene which bestows resistance to a host cell that contains the TSSV against a first toxic agent.
- the first gene can be counter-selected against.
- the recombination cassette, the RecA-like protein gene, and the first gene are linked together on the TSSV such that when the nucleic acid integrates (i.e. resolved) into the particular nucleotide sequence, the RecA-like protein gene and the first gene remain linked together, and neither the RecA-like protein gene nor the first gene remain linked to the integrated nucleic acid.
- the independent origin based cloning vector is a BBPAC and the host cell is a bacterium.
- the BBPAC further contains a second gene that bestows resistance to the host cells against a second toxic agent.
- Introducing the recombination cassette into the host cells is performed by transforming the host cell with the TSSV.
- Inducing the transient expression of the RecA-like protein to support homologous recombination comprises: (i) incubating the host cells at a permissive temperature in the presence of the first toxic agent and the second toxic agent, wherein transformed host cells containing the TSSV and the BBPAC are selected for and wherein the RecA-like protein is expressed.
- a first homologous recombination event occurs between the recombination cassette and the particular nucleotide sequence forming a co-integrate between the TSSV and the BBPAC, wherein the TSSV is either free or part of a co-integrate; (ii) incubating the transformed host cells at a non-permissive temperature in the presence of the first toxic agent and the second toxic agent, wherein host cells containing a TSSV co-integrate are selected for, and wherein free TSSV cannot replicate; (iii) selecting a host cell containing a co-integrate between the TSSV and the BBPAC by Southern analysis; (iv) incubating the host cells containing a co-integrate between the TSSV and the BBPAC at a non-permissive temperature in the presence of the second toxic agent, wherein a second homologous recombination event occurs between the recombination cassette and the particular nucleotide sequence, therein integrating the nucleic acid into the particular nu
- Another embodiment further comprises selecting a host cell containing the resolved BBPAC by colony hybridization with a labeled probe that binds to a DNA homologue of the nucleic acid, an mRNA homologue of the nucleic acid, and/or a protein encoded by the nucleic acid.
- the permissive temperature is 30° C.
- the non-permissive temperature is 43° C.
- the incubating of host cells containing the resolved BBPAC in the presence of the second toxic agent and counter-selecting agent is performed at 37° C.
- Preferred embodiments further comprise the generating of the recombination cassette by placing a first genomic fragment 5' of the specific nucleic acid that is to selectively integrate into the particular nucleotide sequence, and placing a second genomic fragment 3' of the specific nucleic acid.
- the first genomic fragment corresponds to a region of the particular nucleotide sequence that is 5' to the region of the particular nucleotide sequence that corresponds to the second genomic fragment.
- both the first genomic fragment and the second genomic fragment contain 500 or more basepairs of the particular nucleotide sequence.
- the first and second genomic fragments are about the same size.
- both the first genomic fragment and the second genomic fragment contain 1000 or more basepairs of the particular nucleotide sequence.
- the recombination cassette is generated in a building vector and the recombination cassette is subsequently transferred to the TSSV.
- the first gene confers tetracycline resistance and the counter-selecting agent is fusaric acid.
- the RecA-like protein is recA.
- the TSSV is pSV1.RecA having the ATCC no. 97968.
- the RecA-like protein is controlled by an inducible promoter, and the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in the host cell.
- the independent origin based cloning vector is a BBPAC and the recombination deficient host cell is an E. coli bacterium.
- the RecA-like protein is recA.
- the present invention also provides a conditional replication shuttle vector that encodes a RecA-like protein.
- the RecA-like protein is controlled by an inducible promoter.
- the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV).
- the RecA-like protein of the TSSV can be controlled by either a constitutive promoter or by an inducible promoter.
- the TSSV contains a gene that can be counter-selected against.
- the TSSV contains a gene that confers tetracycline resistance.
- the TSSV contains a RecA-like protein that is recA.
- the TSSV contains both a gene that confers tetracycline resistance and a RecA-like protein that is recA.
- the TSSV is pSV1.RecA having the ATCC no. 97968.
- the present invention also provides an independent origin based cloning vector that contains a particular nucleotide sequence that has undergone homologous recombination with a conditional replication shuttle vector in a RecA-host cell, wherein the conditional replication shuttle vector encodes a RecA-like protein.
- the particular nucleotide sequence is part of the gene that encodes the murine zinc finger gene, RU49 which is contained by the independent origin cloning vector.
- the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector in a RecA-host cell, wherein the temperature sensitive shuttle vector encodes a RecA-like protein.
- the independent origin based cloning vector is a BBPAC, and more preferably a BAC.
- the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector that is pSV1.RecA having the ATCC no. 97968.
- the present invention also provides methods of using the modified independent origin based cloning vectors of the present invention to make transgenic animals, perform gene targeting, or perform gene therapy.
- the independent origin based cloning vectors or linearized nucleic acid inserts derived from the IOBCVs, for example, can be introduced into a eukaryotic cell or animal.
- the eukaryotic cell is a fertilized zygote. In another embodiment the eukaryotic cell is a mouse ES cell.
- the gene targeting can be performed to modify a particular gene, or to totally disrupt the gene to form a knockout animal.
- the independent origin based cloning vector contains a nucleic acid that has undergone homologous recombination with a conditional replication shuttle vector in a RecA - whole cell, in which the conditional replication shuttle vector includes a RecA like protein.
- the independent origin based cloning vector is a BBPAC.
- the BBPAC has undergone homologous recombination with a TSSV.
- the BBPAC has undergone homologous recombination with the TSSV that is pSV1.RecA having the ATCC no. 97968.
- One particular embodiment is a method of using the BBPAC to introduce the nucleic acid into an animal to make a transgenic animal comprising pronuclear injecting of the BBPAC (or a linearized nucleic acid insert derived from the BBPAC) into a fertilized zygote.
- the animal is a mammal.
- the mammal is a mouse.
- the independent origin based cloning vector is a BBPAC and the fertilized zygote is a C57BL/6 mouse zygote.
- two picoliters (pl) of less than one ⁇ g/ml BBPAC DNA is injected.
- 2 pl of 0.6 ⁇ g/ml of DNA is injected.
- the present invention also includes a method of using the BBPAC of the invention to perform gene targeting in a vertebrate cells comprising introducing the BBPAC into the vertebrate cell wherein the nucleic acid that has undergone homologous recombination with the conditional shuttle vector, undergoes homologous recombination with the endogenous chromosomal DNA of the vertebrate cell.
- the vertebrate cell is a mammalian cell.
- the mammalian cell is a human cell.
- the vertebrate cell is a fertilized zygote and the nucleic acid contains a disrupted gene.
- the conditional shuttle vector is a TSSV.
- the TSSV is pSV1.RecA having the ATCC no. 97968.
- kits for performing homologous recombination on selected nucleotide sequences contained on an independent origin based cloning vector, such as a BBPAC comprising a conditional replication shuttle vector and a building vector.
- the kit further contains a restriction map for the shuttle vector and/or a restriction map for one or more of the building vectors.
- the kit further includes a protocol for using the contents of the kit to perform homologous recombination.
- kits contains a TSSV, such as pSV1.RecA and a building vector.
- the building vector is pBV.IRES.LacZ.PA.
- the building vector is pBV.EGFP1.
- the building vector is pBV.IRES.EGFP1.
- the building vector is pBV.pGK.Neo.PA.
- kits In a preferred embodiment two or more building vectors are included in the kit. In a more preferred embodiment all four of the above-listed building vectors are included in the kit. Restriction maps for one or more of the building vectors or the TSSV may also be included in the kits. In addition, the kits may also include a protocol for using the contents of the kit to perform homologous recombination. In one specific embodiment, a kit contains pSV1.RecA and one or more of the above-listed vectors also contains fusaric acid and/or chloro-tetracycline.
- FIG. 1 shows the strategy for targeted BAC modification.
- the recombination cassette (genomic fragments A and B; and IRES-LacZP-Poly A marker gene) is first constructed in the building vector and then subcloned into the temperature sensitive pSV1.RecA shuttle vector.
- Co-integrateformation Co-integrates can be formed through homologous recombination at either the homology A or the homology B site, with only the former case illustrated.
- Resolution Resolved BACs are selected by growth on plates containing fusaric acid and chloramphenicol. Correctly resolved clones are identified by colony hybridizations with an insert specific probe (e.g., a PGK polyA probe).
- FIGS. 2A and 2B show a schematic representation of targeted modifications of the BAC 169, which contains the murine zinc finger gene, RU49.
- BAC169 containing RU49 was obtained from screening of the mouse 129 strain BAC genomic DNA library (Research Genetics).
- FIG. 2A depicts a restriction map of the BAC169. The position of several exons are shown. The region of homology A1 (1 kb PCR fragment) and homology B1 (1.6 kb Xba-Hind fragment) are indicated.
- FIG. 1 depicts a restriction map of the BAC169. The position of several exons are shown. The region of homology A1 (1 kb PCR fragment) and homology B1 (1.6 kb Xba-Hind fragment) are indicated.
- 2B depicts a map of the modified BAC169 with IRES LacZ PolyA insertion (BAC169. ILPA). An extra PmeI site is inserted with the marker gene (asterisk). The size of the two Pme-Not fragments and the PmeI fragment are indicated. Since the marker gene (4 kb) is less than the deleted genomic region (7 kb), the total size of the modified BAC (128 kb) is smaller than the original BAC (131 kb).
- FIGS. 3A-3D show Southern blot analyses of BAC co-integrates and resolved BACs.
- FIG. 3A shows a schematic representation of expected Southern blot fragments in BAC169, in co-integrates through homology B1, and in correctly resolved BACs.
- an EcoRI digest is used and homology B1 is used as the probe;
- a HindIII digest is used and the homology A1 is used as probe.
- FIG. 3B shows homology B1 co-integrates.
- the EcoRI digest of BAC clones and controls are probed with homology B1.
- 1-4 represent four clones.
- BAC 169 and pSV1 with the recombination cassette were used as controls.
- FIG. 3C shows the analyses of the 5' ends of the resolved BACs.
- Resolved BAC clones (1-8) were digested with HindIII and probed with homology A1.
- the controls are homology B1 co-integrates (CI), BAC 169 and the shuttle vector with recombination cassettes.
- FIG. 3D shows the analyses of the 3' ends of the resolved BACs. The same procedure is used as described above except the resolved BAC clones were digested with EcoRI and probed with homology B1.
- FIGS. 4A-C show pulsed field gel electrophoresis analyses of modified 169 with the ILPA insertion.
- ILPA (L1 and L2) and BAC169 were prepared by alkaline lysis, and then digested with NotI, PmeI and XhoI (in a standard buffer supplemented with 2.5 mM spermidine). The digested DNA were separated by pulsed field gel electrophoresis (Bio-Rad's CHEF-DRII, 5 to 15s, 15 hours at 14° C.) and blotted on to nitrocellulose filter (Stratagene). The same filter was probed separately with three probes.
- L1 and L2 are lacZ1 and LacZ2 which are independent clones which correspond to clones 1 and 2 respectively in FIGS. 3C and 3D.
- FIG. 4A shows the use of the BAC169 probe which revealed all the restriction fragments.
- FIG. 4B shows the use of the pgkpoly A probe which only hybridized to the ILPA insert fragment.
- FIG. 4C shows the use of the A2 probe which hybridized to a fragment outside the region of modification. The position of the markers are indicated.
- FIGS. 5A-E show the production of BAC transgenic mice.
- FIG. 5A depicts purified linearized BAC L1 128 kb Not I insert for pronuclear injection.
- the pulsed field gel is probed with pgkpolyA probe.
- the numbers represent different fractions.
- the smear below the intact fragment represent degradation and undigested DNA.
- FIG. 5B shows Southern blot analyses of the founder transgenic mice with the lacZ probe.
- the tail DNA were digested with Bam HI and Southern blot analysis was performed.
- the negative control consisted of littermates of Y3, Y7 and Y9 mice.
- the positive control was a conventional transgenic mouse with the lacZ transgene.
- FIGS. 5C and 5D show the results of using PCR to determine the presence of BAC ends in the transgenic mice.
- the DNA at each end corresponding to the vector sequence is amplified and probed with a third oligonucleotide in the middle of the fragment. The appropriate size fragment is indicated.
- the negative controls are littermates.
- the positive control was BAC169 DNA.
- FIG. 5E shows the germline transmission of the lacZ transgene in the Y7 mouse line.
- Tail DNA from two litters having eight mice each were prepared and digested with BamHI. Southern blot analysis was performed with the lacZ probe.
- FIGS. 6A-D show the expression of the lacZ transgene in the brain of the Y7 BAC transgenic line.
- P6 mice brain from Y7 transgenic mice (FIG. 6A) and a wild type control litter mate (FIG. 6B) were whole mount stained to reveal lacZ expression in the Y7 cerebellum. Thick saggital sections (5 mm) from Y7 transgenic mice were also stained for lacZ expression.
- FIG. 6C shows the low magnification and FIG. 6D shows the high magnification of the rectangle area indicated in FIG. 6C. Expression in the cerebellum, the detate gyrus and the lineage of the olfactory bulb are indicated (i.e. SVZ, RMS and the OB).
- Ce cerebellum
- SC superior collicoli
- IC inferior colliculi
- DG dentate gyrus
- VZ ventricular zone
- SVZ subventricular zone
- LV lateral ventricle
- RMS rostral migratory tract
- OB olfactory bulb
- Co cortex
- FIGS. 7A-C are schematic diagrams containing a hypothetical map of a gene of interest within a selected BAC; the first targeted modification to introduce the positive selection marker gene; and the second modification to delete the promoter of the gene and to generate the short arm.
- FIG. 8 is the restriction map of pSV1.RecA. This temperature sensitive shuttle vector is based on the pMBO96 vector originally constructed by M. O'Connor et al. [Science, 244:1307-1312 (1989)].
- FIG. 9 is the restriction map of pBV.IRES.LacZ.PA. This vector was modified from the pWH10 vector originally constructed by Kim et al. [MCB, 12:3636-3643 (1992)].
- FIG. 10 is the restriction map of pBV.EGFP1.
- the plasmid is based on pBluescript.KS(+).
- EGFP1 was from Clonetech.
- FIG. 11 is the restriction map of pBV.IRES.EGFP1.
- the plasmid is based on the pBluescript.KS back bone.
- EGFP1 was from Clonetech.
- FIG. 12 is the restriction map of pBV.PGK.Neo.PA.
- the vector is based on a pBS.KS backbone.
- the pGK.Neo.PA sequences was excised from a pKS.NT vector by digestion with HindIII and BamHI and subcloned into the HindIII/Bam fragment of the BV.IRES.LacA.PA.
- the present invention provides a simple method for directly modifying an independent origin based cloning vector (IOBCV) in recombination deficient host cells including generating deletions, substitutions, and/or point mutations in a specific gene contained in the independent origin based cloning vector. Such modifications may be performed with great specificity.
- the modified independent origin based cloning vectors of the present invention can be used to introduce a modified heterologous gene into a host cell.
- One specific use of such a modified vector is for the production of a germline transmitted independent origin based cloning vector transgenic animal.
- Targeted independent origin based cloning vector modification can be used for functional studies in diverse biological systems.
- the ability to efficiently modify a independent origin based cloning vector and generate an IOBCV-transgenic animal has important applications for functional analyses of genes in vivo.
- modified independent origin based cloning vectors can be used to study regulation of genes or gene complexes in transgenic animals such as mice. Since modified independent origin based cloning vectors can be used to study gene function in vivo, a deletion, substitution and point mutation within a given gene can be made in a independent origin based cloning vector, and the independent origin based cloning vector containing the modified gene can be reintroduced in vivo in its endogenous expression pattern.
- targeted independent origin based cloning vector modification can be used to create targeted expression of a selected gene, in the expression pattern of another gene, without prior knowledge of all of the regulatory elements of the selected gene.
- An important application of this type is targeted expression of the cre recominase for tissue/cell type specific gene targeting [Kuhn et al., Science 269:1427 (1995); Tsien et al., Cell 87:1317 (1996)].
- modified independent origin based cloning vectors can be used to generate large DNA constructs particularly for gene targeting in ES cells and in vivo.
- the independent origin based cloning vector is a Bacterial Artificial Chromosome (BAC) modified in a host E. coli cell.
- BAC Bacterial Artificial Chromosome
- a targeted BAC modification system has several advantages over a conventional yeast based modification system. First, a modified BAC automatically returns to the recombination deficient state after modification, ensuring stable maintenance of the modified BAC in the host strain. Second, BAC DNA can be very easily purified in relatively large quantities and high quality, allowing for use in biological experimentation including pronuclear injection.
- BAC libraries there are many more BAC libraries available from different species of animal, plants and microbes [Woo et al., Nucleic Acids Res., 22:4922 (1994); Wang et al., Genomics 24:527 (1994); Wooster et al., Nature 378:789 (1995)]. Most BACs also include all the necessary regulatory elements (i.e. LCRs and enhancers) to obtain dose dependent and integration site independent transgene expression [Dillon et al. Trends Genet. 9:134 (1993); Wilson et al., Annu. Rev. Cell. Biol. 6:679 (1990); Bradley et al., Nature Genet.
- LCRs and enhancers i.e. LCRs and enhancers
- Targeted BAC modification can be applied successively to dissect these elements.
- a modified BAC may be used to generate a transgenic animal.
- the BAC (or PAC) stably integrates into the animal cell genome.
- the transgenic animal can be used for functional studies, or for generating a desired gene product, such as producing a human protein in the milk of a transgenic mammal [Drohan et al. U.S. Pat. No. 5,589,604, Issued Dec. 31, 1996].
- modified BACs or PACs may be used for delivering a specific gene in gene therapy.
- a modified BAC has been successfully inserted into a murine subject animal, and in vivo heterologous gene expression has been demonstrated.
- the methodology of the present invention is very general. Whereas the targeted independent origin based cloning vector modification is demonstrated on BACs, the system is readily applicable to BBPACs in general including PACs, P1 and other vectors propagated in the recombination deficient E. coli.
- the BAC modification exemplified herein is also apropo to Mammalian Artificial Chromosomes. For example, Harrington et al. [Nature Genetics, 15:345-355 (1997)] have used BAC derived DNA as a component of their Human Artificial Chromosome. Therefore, the use of such human artificial chromosomes can include the BAC modification taught by the present invention.
- an "IOBCV” is an independent origin based cloning vector.
- One example of such a cloning vector is a BBPAC defined below.
- An IOBCV generally comprises a nucleic acid insert which either is or contains a gene of interest.
- a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
- a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in viva, i.e., capable of replication under its own control.
- a "Bacterial or Bacteriophage-Derived Artificial Chromosome” or “BBPAC” denotes a vector that is derived from a bacterium or bacteriophage such as a Bacterial Artificial Chromosome (BAC) which is an E. coli F element based cloning system, a P1-Derived Artificial Chromosome (PAC) or a lambda-based cosmid.
- the BBPAC encodes up to 500 kilobases of genomic sequences.
- the BBPAC encodes between 120 to 180 kilobases of genomic sequences.
- the BBPAC encodes 130 kilobases of genomic sequences.
- a BBPAC used for gene targeting can be referred to as a "BBPAC targeting construct" and contains a nucleic acid insert comprising the gene targeting construct.
- a “gene targeting construct” as used herein is used interchangeably with “targeting construct” and is a nucleic acid that when introduced into a cell undergoes homologous recombination with the endogenous chromosomal DNA of the cell.
- the nucleic acid is introduced into the cell to induce a modification of a particular gene contained on the endogenous chromosomal DNA, including in particular cases, to disrupt that gene to create a knockout animal.
- a recombinant deficient host cell is "RecA -" when the host cell is unable to express a RecA-like protein, including recA itself, which can support homologous recombination.
- the gene encoding the RecA-like protein has been deleted in a RecA - host cell.
- the RecA-host cell contains a mutation in the recA gene that impairs its function.
- RecA-like protein is defined herein to have the meaning generally accepted in the art except as used herein the recA protein itself is included as being a specific RecA-like protein.
- RecA-like proteins are proteins involved in homologous recombination and are homologs to recA [Clark et al., Critical Reviews in Microbiology 20:125-142 (1994)].
- the recA protein is the central enzyme in prokaryotic homologous recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination [McKee et al., Chromosoma 7:479-488 (1996)].
- RecA-like proteins have been found in eukaryotic organisms and yeast [Reiss et al., Proc. Natl. Acad. Sci. 93:3094-3098 (1996)].
- Two RecA-like proteins in yeast are Rad51 and Dmc1 [McKee et al. (1996) supra].
- Rad51 is a highly conserved RecA-like protein in eukaryotes [Peakman et al., Proc. Natl. Acad. Sci. 93:10222-10227 (1996)].
- a "gene of interest” is a gene contained by a host cell genome or more preferably an independent origin based cloning vector that has been selected to undergo homologous recombination with a specific nucleic acid contained in a recombination cassette.
- a gene of interest can be either specifically placed into the host cell or independent origin based cloning vector for this purpose, or already contained by the host cell or independent origin based cloning vector .
- a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
- the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
- the present invention provides a recombination cassette that includes two homology fragments interrupted by an insertion, deletion or mutation sequence.
- Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
- the heterologous DNA includes a gene foreign to the cell.
- nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
- nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
- this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
- sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
- a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
- Homologous recombination refers to the insertion of a modified or foreign DNA sequence contained by a first vector into another DNA sequence contained in second vector, or a chromosome of a cell.
- the first vector targets a specific chromosomal site for homologous recombination.
- the first vector will contain sufficiently long regions of homology to sequences of the second vector or chromosome to allow complementary binding and incorporation of DNA from the first vector into the DNA of the second vector, or the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
- a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
- a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended or expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
- Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
- polyadenylation signals are control sequences.
- a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
- a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
- a “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
- the term "translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.
- a particular nucleotide sequence comprising a gene of interest can be isolated from any source, particularly from a human cDNA or genomic library.
- methods well known in the art, as described above can be used for obtaining such genes from any source (see, e.g., Sambrook et al., 1989, supra).
- any animal cell potentially can serve as the nucleic acid source for the molecular cloning of any selected gene.
- the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. 1, 11).
- Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences.
- the present invention provides methods for selectively performing homologous recombination in a cell that normally cannot independently support homologous recombination.
- a specific nucleic acid is inserted into a recombination cassette that selectively integrates into a particular nucleotide sequence when the recombination deficient cell is transiently induced to support homologous recombination.
- the present invention allows the integration of a specific nucleic acid into a particular nucleotide sequence of a gene of interest.
- the methods provided by the present invention minimize the nonspecific nucleotide sequence rearrangements and deletions, which are characteristic of other systems which involve host cells that normally support homologous recombination.
- the specific nucleic acid can encode an entirely different protein than the gene of interest, and the gene of interest may be selected for the tissue specificity of its promoter, for example for use in generating a transgenic animal, or in a gene therapy protocol.
- the rat preproenkephalin gene may be used as the gene of interest since the preproenkephalin promoter has been shown to confer brain expression and synaptic regulation in transgenic mice. [Donovan et al., Proc. Natl. Acad. Sci. 89:2345-2349 (1992)].
- the murine zinc finger gene, RU49 was used as the gene of interest.
- the specific nucleic acid can be constructed so as to cause a deliberate and specific modification in the sequence of the gene of interest, for example for inducing a change in the amino acid sequence of the gene product, such as is typically done in site-directed mutagenesis protocols.
- the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA - .
- alternative causes for recombination deficiency may be rectified by methods that are analogous to those taught by the present invention and/or readily apparent in view of such teachings.
- recombination deficiency may be due to a deficiency of an alternative recombination protein such as another Rec protein including recB, recC, recD, and recE [Clark et al., Critical Reviews in Microbiol. 20:125-142 (1994)] which may be manipulated in a manner that is analogous to that taught herein for RecA-like proteins.
- inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of a RecA-like protein in the host cell.
- Such induction may be performed by expressing a RecA-like protein contained by the recombination deficient host that is under the control of an inducible promoter.
- conditional replication shuttle vectors can also include pBR322 in a polyA temperature-sensitive bacterial strain.
- the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
- TSSV temperature sensitive shuttle vector
- Inducing the transient expression of the RecA-like protein consists of transforming the host cell with the TSSV at a permissive temperature, and growing the host cell at a non-permissive temperature.
- the TSSV encodes a RecA-like protein that is expressed in the host cell and supports the homologous recombination between a specific nucleic acid contained in a recombination cassette and the particular nucleotide sequence contained in the host cell.
- the TSSV encoding the RecA-like protein is diluted out when the host cell is grown at the non-permissive temperature.
- the particular nucleotide sequence which has been selected to undergo homologous recombination is contained by an independent origin based cloning vector (IOBCV) that is comprised by the host cell, and neither the independent origin based cloning vector alone, nor the independent origin based cloning vector in combination with the host cell, can independently support homologous recombination.
- IBCV independent origin based cloning vector
- both the independent origin based cloning vector and the host cell are RecA - , and inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of the RecA-like protein to support homologous recombination in the host cell.
- the independent origin based cloning vector can be a BBPAC, such as the BAC exemplified below and the host cell can be a host bacterium, such as E. coli.
- the independent origin based cloning vectors for use in the methods of the present invention can be obtained from a number of sources.
- E. coli-based artificial chromosomes for human libraries have been described [Shizuya et al., Proc. Natl. Acad. Sci. 89:8794-8797 (1992); Vietnamese et al., In Current Protocols in Human Genetics (ed. Dracopoli et al.) 5.15.1-5.15.24 John Wiley & Sons, New York (1996); Kim et al., Genomics 34:213-218 (1996)].
- BAC, PAC, and P1 libraries are also available for a variety of species (e.g. Research Genetics, Inc., Genome Research, Inc., Texas A&M has a BAC center to make a BAC library for livestock and important crops). Also BACs can be used as a component of mammalian artificial chromosomes.
- An independent origin based cloning vector that is a BAC can be isolated using a cDNA or genomic DNA probe to screen a BAC genomic DNA library, for example.
- the use of a mouse genomic BAC library from Research Genetics is exemplified below.
- a positive BAC can generally be obtained in a few days.
- To insert a gene of interest into a selected locus in the BAC the region of insertion can be mapped for restriction enzyme sites. Whereas subcloning is necessary for detailed mapping, it is generally unnecessary since rough mapping is usually sufficient.
- other independent origin based cloning vector genomic libraries can be screened and the isolated independent origin based cloning vectors manipulated in an analogous fashion.
- conditional replication shuttle vectors of the present invention are constructed so as to contain a recombination cassette that can selectively integrate into the nucleotide sequence of the gene of interest encoded by the independent origin based cloning vector.
- Such conditional replication shuttle vectors can be constructed by inserting a PCR amplified RecA-like gene into an appropriate conditional replication shuttle vector which either contains a specific drug resistant gene or can be subsequently modified to contain one.
- the drug resistant gene can also be counter-selected against, such as with, tetracycline and fusaric acid.
- the conditional shuttle vector can also contain a counter-selection gene such as a gene that confers sensitivity to galactose, for example.
- the E. coli K12 recA gene (1.3 kb) is inserted into the BamHI site of a pMBO96 vector.
- the vector already carried a gene that bestows tetracycline resistance, and in addition contains a pSC101 temperature sensitive origin of replication, which allows the plasmid to replicate at 30 degrees but not at 43 degrees celsius.
- the RecA-like protein of a conditional replication shuttle vector can be controlled by either an inducible promoter or a constitutive promoter.
- the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in a host cell.
- the constitutive promoter is the endogenous E. coli recA promoter.
- the conditional replication shuttle vector should also contain at least one unique cloning site.
- one unique site is reserved for transferring the recombination cassette containing the specific nucleic acid from the building vector to the conditional replication shuttle vector.
- a polylinker can be inserted between two specific restriction sites to create additional restriction sites that allow cloning of the recombination cassette into the conditional replication shuttle vector.
- conditional replication shuttle vector created should minimally contain a recombination cassette comprising the specific nucleic acid, (e.g., containing a point mutation, deletion or a marker gene) flanked at both the 5' and 3' ends by genomic fragments containing 400 basepairs or more of the gene of interest of the independent origin based cloning vector.
- specific nucleic acid e.g., containing a point mutation, deletion or a marker gene
- a building vector is used to construct the recombination cassette.
- Two small genomic fragments, each containing about 500 basepairs (400 basepairs to 600 basepairs is sufficient) of the gene of interest are cloned into the building vector (e.g., pBV1) in appropriate order and orientation to generate the flanking regions of the recombination cassette.
- the recombination cassette is then transferred into the conditional replication shuttle vector (e.g., pSV1.RecA).
- conditional replication shuttle vector is a TSSV and the TSSV is pSV1.RecA having the ATCC no. 97968, which has been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20110-2209, on Mar. 25, 1997 under the Budapest Treaty.
- conditional replication shuttle vector is transformed into a RecA - host cell containing the independent origin based cloning vector.
- the independent origin based cloning vector can also contain a gene which bestows resistance to a host cell against a corresponding toxic agent/drug such as an antibiotic or in a specific embodiment, chloramphenicol.
- conditional replication shuttle vector can replicate (e.g., when the conditional replication shuttle vector is a TSSV which replicates at 30° but not at 43°, the host cell is grown at 30° C.) and the transformants can be selected via the specific drug resistant gene (or first drug resistant gene) carried by conditional replication shuttle vector, and the second drug resistant gene carried by the independent origin based cloning vector. Since the conditional replication shuttle vector also carries the RecA-like protein gene, homologous recombination can occur between the conditional replication shuttle vector and the independent origin based cloning vector to form co-integrates through the sequence homology at either the 5' or the 3' flanking regions of the recombination cassette.
- the co-integrates then can be selected by growing the cells on plates containing the first and second drugs at non-permissive conditions (e.g. for the TSSV above, at 43° C.) so that the non-integrated, free conditional replication shuttle vectors are lost. This results in the selection for host cells carrying the integrated conditional replication shuttle vectors, (which co-integrate either into the independent origin based cloning vector or into the host chromosome). Correct independent origin based cloning vector co-integrates can be identified by PCR or more preferably with Southern blot analyses.
- the co-integrates can then be re-streaked onto plates containing the second drug, (i.e., the drug which the gene initially carried by the independent origin based cloning vector protects against) and grown under non-permissive conditions overnight.
- a fraction of the co-integrates undergo a second recombination event (defined as resolution), through sequence homology at either the 5' or the 3' flanking regions of the recombination cassette.
- the resolved independent origin based cloning vector automatically loses both the first drug resistant gene (i.e., the specific drug resistant gene contained by the conditional replication shuttle vector) and the RecA-like protein gene due to the linkage arrangement of the RecA-like protein gene, the drug resistant gene and the specific nucleic acid on the conditional replication shuttle vector, described above.
- the excised conditional replication shuttle vector cannot replicate under the non-permissive conditions and is therefore diluted out.
- the resolved independent origin based cloning vectors can be further selected for by growing the host cells (e.g., at 37° C.) on plates containing the second drug and an agent that counterselects against cells containing the gene resistant to the first drug (e.g., a gene conferring tetracycline resistance may be counter-selected against with fusaric acid).
- the resolved independent origin based cloning vector will be either the original independent origin based cloning vector or the precisely modified independent origin based cloning vector.
- One method to identify the correctly resolved BAC is to choose 5-10 colonies and prepare a miniprep DNA. The DNA can then be analyzed using Southern blots to detect the correct targeting events.
- the desired clones can be identified by colony hybridization using a labeled probe for the specific nucleic acid contained by the recombination cassette.
- probes are well known in the art, and include labeled nucleotides probes that hybridize to the nucleic acid sequence.
- a marker nucleic acid can be included in the recombination cassette and constructed so as to remain with the specific nucleic acid upon integration into the independent origin based cloning vector.
- the marker nucleic acid can encode a protein that confers a specific drug resistance to the host cell, a protein that confers a particular physical characteristic to the cells, such as a green fluorescent protein, or it can be any other marker protein including e.g., ⁇ -galactosidase.
- the methods of homologous recombination of the present invention are selective, and nonspecific nucleotide sequence rearrangements either do not occur, or arc essentially undetectable by one or more conventional methods of analysis.
- One such method includes pulsed field gel mapping of the modified independent origin based cloning vector and the unmodified independent origin based cloning vector to determine whether any unexpected deletions, or insertions or rearrangement were generated during the modification procedure.
- the same filter can be probed separately with a probe for the whole independent origin based cloning vector, with a probe for the specific nucleic acid, and a probe for a region of the gene of interest that has not been modified.
- a restriction enzyme digestion can reveal a finger print of the modified independent origin based cloning vectors indicating whether the fragments are preserved. Such a restriction enzyme digestion is exemplified below. Restriction enzyme digestions can be repeated with one or more additional restriction enzymes selected with respect to the restriction site map of the independent origin based cloning vector.
- the modified independent origin based cloning vector and the unmodified independent origin based cloning vector can be assayed with both a probe specific for any region of the DNA contained by the recombination cassette predicted to be inserted into the independent origin based cloning vector (e.g., the promoter sequence, the specific nucleic acid, and a polyadenine addition signal sequence) and a probe specific for a region outside of the modification region (e.g., near the promoter region but outside of the modification region).
- a probe specific for any region of the DNA contained by the recombination cassette predicted to be inserted into the independent origin based cloning vector e.g., the promoter sequence, the specific nucleic acid, and a polyadenine addition signal sequence
- a probe specific for a region outside of the modification region e.g., near the promoter region but outside of the modification region.
- a modified independent origin based cloning vector of the present invention can be purified by gel filtration, e.g. a column filled with SEPHAROSE CL-4B yielded intact linear BAC DNA.
- the column can be pre-equilibrated in an appropriate buffer, as described in the Example below.
- the purified DNA can be directly visualized with ultraviolet light after ethidium bromide staining, for example.
- Columns such as the SEPHAROSE CL-4B column also can efficiently separate degraded DNA from the pure linear DNA.
- the present invention also provides methods of using the modified independent origin based cloning vectors of the present invention.
- modified independent origin based cloning vectors contain a nucleic acid that can be inserted into an animal to make a transgenic animal.
- the modified independent origin based cloning vectors of the present invention can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).
- Various therapeutic heterologous genes can be inserted into an independent origin based cloning vector of the invention such as but not limited to adenosine deaminase (ADA) to treat severe combined immunodeficiency (SCID); marker genes or lymphokine genes into tumor infiltrating (TIL) T cells [Kasis et al., Proc. Natl. Acad. Sci. U.S.A. 87:473 (1990); Culver et al., ibid. 88:3155 (1991)]; genes for clotting factors such as Factor VIII and Factor IX for treating hemophilia [Dwarki et al.
- ADA adenosine deaminase
- SCID severe combined immunodeficiency
- marker genes or lymphokine genes into tumor infiltrating (TIL) T cells [Kasis et al., Proc. Natl. Acad. Sci. U.S.A. 87:473 (1990);
- One particular method comprises the pronuclear injection of the modified independent origin based cloning vector into a fertilized animal zygote.
- a method is exemplified below with the modified independent origin based cloning vector being a BAC which has been linearized, and the animal zygote being a mouse zygote. 2 pl of 0.6 ⁇ g/ml of BAC DNA was injected.
- the presence of both ends of the modified independent origin based cloning vector can be assayed for in the transgenic animal to determine if the intact nucleic acid insert of the IOBCV has been integrated into the genome. Since both ends of the nucleic acid insert contain some vector sequence, PCR primers specific to the vector sequence can be generated and used to amplify the transgenic DNA. The amplified products can then be probed with a third labeled oligonucleotide probe within the amplified region.
- transgenic animals that are formed give rise to germline transmission after appropriate breeding (B6/CBA mice were used in the Example).
- the ratio of transgenic animals to wild type animals should follow Mendelian genetics.
- the expression of the specific nucleic acid and/or the gene of interest inserted into the transgenic animal can be determined by a variety of methods well known in the art which depend on the nature of the insert. For example, enzymes can be appropriately assayed for activity, in the case of ⁇ -galactosidase, whole mount staining can be performed, in situ hybridization can be used to detect the corresponding mRNA, and specific antibodies can be used to identify the expression of a corresponding protein. In preferred embodiments such expression will be evident only in cells in which the endogenous gene of interest is expressed.
- the present invention also provides the use of targeted BBPAC modification to obtain a high rate of gene targeting in vertebrates.
- the BBPAC contains a nucleic acid insert comprising the gene targeting construct.
- the circular BBPAC can be used, or preferably the linearized nucleic acid insert is used. In either case, the BBPAC or linearized nucleic acid insert can be purified by gel filtration as described herein.
- the gene targeting is performed in ES cells using a BBPAC gene targeting construct that is greater than 100 kb.
- the BBPAC gene targeting construct is similar to the conventional positive selection gene targeting construct (FIG. 7): it contains two regions of homology, a long arm (>80 kb) and a short arm (10-20 kb), with the neo cassette (pgk-neo-polyA) introduced into the middle of the BBPAC.
- Two targeted BBPAC modifications are used to make this construct. The first modification is to introduce the neo gene to disrupt the gene of interest in the BBPAC. A second modification is to create the short arm (10-20 kb).
- the reason for the second modification is enable the use of an endogenous probe flanking the short arm (KO probe) to detect a polymorphism between the targeted allele and the wild type allele in screening ES cell clones (FIG. 7; Gene Targeting, a practical approach, supra).
- a preferred version of the BBPAC gene targeting methodology of the present invention also includes negative selection.
- the conventional negative selection cassettes such as the use of the herpes thymidine kinase cassette or the diphtheria toxin gene cassette, may not always work with BBPAC constructs since BBPAC DNA tends to exist in transfected mammalian cells as episomal DNA for a long period of time [Baker et al., NAR 25:1950-1956].
- the EGFP1 cassette can be used as a negative screening cassette.
- the CMV promoter driven green fluorescent protein (EGFP-1) and the polyA signal can be introduced.
- GFP is not toxic to the cells but serves as a fluorescent marker protein.
- the EGFP-1 cassette When gene targeting occurs, the EGFP-1 cassette will be lost and the cell will not exhibit a green fluorescence under UV light.
- the BBPAC integrates non-homologously, the EGFP-1 cassette also integrates, and the cells will therefore exhibit the green fluorescence under UV.
- the definitive Southern blot analyses only those neo resistant cell lines which do not exhibit a green fluorescence under UV light are chosen.
- the process of generating the targeted ES cells with a BBPAC targeting construct is essentially the same as with the conventional protocols (Gene Targeting, A Practical Approach, supra), except for the following steps.
- the transfection of ES cells with the linearized intact BBPAC nucliec acid insert is performed as described by Baker [NAR, 25:1950-1956 (1997)], using psoralen-inactivated adenovirus as carriers, for example.
- the method enables transfection efficiency in mammalian cells with linear BBPAC DNA to be similar to the transfection efficiency of a conventional DNA construct.
- the BBPAC targeting construct can potentially provide 10-100 fold higher targeting frequency than the conventional targeting construct, thereby making gene targeting in mouse ES cells easier and cheaper, since only a few dozen colonies need to be isolated and screened to obtain the targeted clones.
- the present invention further provides a method of performing gene targeting in fertilized vertebrate zygotes by the injection of a BBPAC targeting construct, or preferably the linearized intact BBPAC nucleic acid insert containing the targeting construct to generate a transgenic knock-out animal (TKO).
- a large targeting construct >100 kb
- TKO methodology has previously been attempted by Brinster et al.
- the design of the gene targeting construct is similar to the ES cell targeting construct except that instead of the neo gene, an IRES-GFP cassette or an IRES-lacZ cassette is fused to an exon of the gene of interest to disrupt the gene (FIG. 7).
- an IRES-GFP cassette or an IRES-lacZ cassette is fused to an exon of the gene of interest to disrupt the gene (FIG. 7).
- two consecutive steps of BBPAC modifications are involved in generating the BBPAC containing the gene targeting construct.
- the modified BBPAC TKO construct can be prepared in milligram quantities and linearized as described above.
- the linearized DNA then is introduced into the fertilized zygote by a standard protocol, e.g., pronuclear injection (Hogan et al., (1986) supra).
- the transgenic animal is then identified by standard Southern blots.
- the gene targeting event can be further identified by digesting DNA of the transgenic animal with appropriate enzymes, such as enzyme X, (FIG. 7) and probed with the flanking KO probe (FIG. 7). Mice with the targeting event will have an additional band of the appropriate size.
- Such gene targeting events can further be confirmed by expression of the GFP or LacZ marker gene in the expression pattern of the targeted endogenous gene, since the construct is designed to trap the endogenous promoter.
- the TKO method has important ramifications in the field of vertebrate genetics. It enables gene targeting in many organisms that do not have ES cells, such as zebra fish, rats and other mammals. This will help to generate better animal models for human diseases (e.g., rats and monkeys), or to create genetically targeted animals suitable for organ transplants (such as pigs or baboons) or for commercial reasons (e.g., leaner pork or beef).
- This method also has additional advantages, even for gene targeting in mice. For example, this method will automatically provide germline transmission, since transgenic animals are rarely chimeric. It also enables targeted mice in strains other than the 129 strain to be obtained, and avoids the expensive and time-consuming out-breeding protocols.
- BBPAC targeting constructs are provided. Since gene targeting in somatic cells is also dependent on the length of homology, using large DNA targeting construct also improves the targeting rate in somatic cells.
- the experimental design in this case is similar to that with the ES cells described above. Somatic cell gene targeting is useful in gene therapy, for example, in a targeted insertion of a functional gene in a hereditary disease of the hematopoietic system. Such methods are also useful to generate targeted cell lines for experimental purposes.
- conditional replication shuttle vectors that encode a RecA-like protein are also provided by the present invention.
- the RecA-like protein can be controlled by either an inducible promoter or a constitutive promoter.
- the conditional replication shuttle vector is preferably a temperature sensitive shuttle vector (TSSV).
- TSSV temperature sensitive shuttle vector
- the TSSV contains both a gene that confers tetracycline resistance and a RecA-like protein that is recA.
- the TSSV is pSV1.RecA having the ATCC no. 97968.
- Independent origin based cloning vectors that contain a gene of interest that has been modified by the methods of the present invention are also included in the present invention. More particularly such independent origin based cloning vectors have undergone homologous recombination with a conditional replication shuttle vector in a RecA - host cell, wherein the conditional replication shuttle vector encodes a RecA-like protein.
- the independent origin based cloning vector has undergone homologous recombination in a RecA - host cell with a temperature sensitive shuttle vector encoding a RecA-like protein.
- the modified independent origin based cloning vector is a BAC that has undergone homologous recombination with the temperature sensitive shuttle vector pSV1.RecA having the ATCC no. 97968.
- Bacterial based artificial chromosomes such as Bacterial artificial chromosomes (BACs) and P-1derived artificial chromosomes (PACs), are circular bacterial plasmids that may propogate as large as 300 kb of exogenous genomic DNA (Shizuya et al, PNAS, 89, 8794-97, 1992; loannou et al, Nature Genet., 6, 84-90, 1994). For the majority of BAC and PAC libraries, the average size of the insert is 130-150 kb.
- BACs Bacterial artificial chromosomes
- PACs P-1derived artificial chromosomes
- BAC and PAC libraries are much easier to construct due to higher cloning efficiency.
- Second, BACs and PACs are propagated in recombination deficient E. coli host cells, so they have high stability and minimal chimerism. No rearrangements have been observed in BACs or PACs after 100 generations of growth.
- Third, isolation of BAC and PAC DNA is very easy since they exist as supercoiled circular plasmids that are resistant to shearing. Conventional bacterial plasmid DNA isolation methods can be applied to obtain milligrams of intact BAC or PAC DNA.
- Third, direct DNA sequencing can be applied to BAC or PAC DNA, which is not possible for YAC DNA.
- BBPACs are useful for physical mapping in genome studies, no simple method is available to modify BBPACs, as is available for the YACs.
- a simple homologous recombination based BBPAC modification method is disclosed, termed targeted BBPAC modification (See FIG. 7 for a schematic representation of the method). This method allows precise modification, such as marker insertion, deletion, point mutation, at any chosen site within a given BBPAC.
- This method involves several steps: isolation of BBPACs using cDNA or genomic DNA probes, simple mapping and partial sequencing of the BBPACs, cloning of the shuttle vector, targeted modifications, pulsed field gel analyses of the modified BBPACs, and finally preparation of linearized BBPAC DNA for functional studies, such as pronuclear injection to produce BBPAC transgenic mice. Since the method is simple and reliable, it is reasonable to expect that the entire procedure, from the step of screening for a BBPAC with a cDNA or genomic DNA probe to the step of modified BBPACs ready for functional studies, can be completed within 6-8 weeks.
- the IRES-LacZ marker gene has been introduced into an 131 kb bacterial artificial chromosome (BAC) containing the murine zinc finger gene, RU49. No rearrangements or deletions are detected in the modified BACs. Furthermore, transgenic mice are generated by pronuclear injection of the modified BAC and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the cerebellum has been observed, which could not be obtained with conventional transgenic constructs. In summary, a novel and efficient method has been developed to modify BACs, PACs and P1 for in vivo studies of gene expression and gene function.
- BAC 131 kb bacterial artificial chromosome
- a BAC clone is isolated with either a unique cDNA or genomic DNA probe.
- BAC libraries for various species (in the form of high density BAC colony DNA membrane) are available from Research Genetics, Inc. and Genome Research, Inc.
- the mouse 129 genomic BAC library from Research Genetics has proved to be a good source for genomic DNAs.
- the probe is first tested on a mouse genomic Southern blot to ensure that the probe does not contain any repetitive elements.
- the library is screened according to manufacture's direction. The positive clones can be obtained from the company within a few days.
- Solution I 50 mM glucose, 25 mM Tris.HCl (pH 8.0); 10 mM EDTA (pH 8.0)
- Solution II 0.2N NaOH, 1% SDS (0.4 g NaOH, 45 ml ddH2O, 5 ml 10% SDS).
- Solution III 5M KOAc (60 ml), glacial acetic acid (11.5 ml), H2O (28.5 ml).
- the BAC midiprep DNA may be stored in 4° C. for months (Do not freeze the BAC DNA, since repetitive freezing and thawing will result in degradations).
- the first method is the standard cesium chloride banding method (see Maniatis, supra). This method was used routinely to obtain >500 ug BAC DNA from 1 liter bacteria culture.
- the second method uses a commercially available column, the Nucleobond AX-500 (made by The Nest Group, Southborough, Mass.). The maxiprep DNA are also stored in 4° C. for long-term storage.
- a simple mapping of the BACs is done.
- the following enzymes are used to map each BAC: Not I (to release the BAC insert), Mlu I, NotI/Mlu I (double digest), PmeI, PmeI/NotI and XhoI.
- Digestion is done in a 40 ul total volume, which contains the following: 5 ul midiprep DNA, 4 ul digestion buffer, 4 ul 10 ⁇ BSA (if necessary), 1 ul 100 mM spermidine (final concentration 2.5 mM), 2 ul enzyme (10-40 units), and ddH2O. Digestion is done at 37° C. for >5 hrs.
- the digested BACs are resolved on a pulsed field gel (Bio-Rad's CHEF-DRII).
- the gel is 1% agarose in 0.5 ⁇ TBE.
- the gel is run in 0.5 ⁇ TBE.
- the separation condition is the following: 6v/cm, 5s to 15s linear ramping for 15 hrs to 18 hrs at 14° C.
- the New England Biolab's PFGE marker I or II as the high molecular weight marker and 1 kb DNA ladder (Life Technologies Inc.) as the low molecular weight marker are used.
- the gel is then stained with ethidium bromide (1 to 5000, or 1 to 10,000 dilution of 10 mg/ml stock) for 30 min prior to taking the photograph. Then the gel is blotted onto the nitrocellulose membrane and hybridized to cDNA and genomic DNA probes according to standard protocols (Maniatis, supra). To ensure the entire cDNA is included in the BAC, probes/or oligonucleotides from both the 5' end and the 3' end of the gene are used to probe the blot separately. Those large BACs containing the entire gene are usually selected for BAC modification.
- targeted BAC modification is a method based on homologous recombination
- homologous sequence from the BAC has to be obtained.
- Two homologous sequences of about 500 bp each (namely A and B, FIG. 7) is all that is needed to construct the shuttle vector for BAC modification.
- the homologous sequences are chosen such that a given modification (i.e. insertion, deletion and point mutation) will be introduced between A and B in the BAC.
- a and B can be obtained by direct sequencing of the BACs.
- the sequencing oligonucleotides are designed based on the cDNA sequence.
- step 2 If maxiprep DNA is used, go directly to step 2. If midiprep DNA is used, first add 100 ul ddH2O and 10 ul 10 mg/ml RNAse A to 100 ul midiprep BAC DNA, and incubate at 37° C. for >1 hr. (This step is critical, incomplete RNAse treatment will result in poor precipitation and sequencing).
- Each sequencing reaction will result in up to a 500 bp sequence. Sequence more than one BAC for a given primer to compare the sequences.
- the main purpose for sequencing is to design a 20 bp PCR primer, which is about 500 bp away from the sequencing oligo (which usually is the other PCR primer), to enable PCR amplification of this genomic fragment and to clone it into the building vector. Therefore, as long as a 20 bp sequence can be identified which is at the appropriate position, and which is the same in several independent sequencing reactions, the goal is achieved.
- the quality of the DNA sequence in between is not very critical.
- a two vector system is designed to construct the shuttle vector for BAC modification (FIG. 1).
- the first vector is a pBS.KS based building vector, which is used to construct the recombination cassette containing homologous sequence A and homologous sequence B and the modification to be introduced between them.
- the recombination cassette was not constructed in the pSV1.
- RecA shuttle vector was for the following reasons: first, it is a low copy plasmid so that it is difficult to obtain high quantity DNA; second, it is a large plasmid (11 kb), so it is relatively difficult to clone.
- the building vector contains the marker gene to be introduced into the BAC, cloning sites flanking it (usually EcoRI for cloning the homology A and XbaI for homology B, and rare restriction sites such as MluI, PmeI and Pac I for mapping of the modified BAC).
- cloning sites flanking it usually EcoRI for cloning the homology A and XbaI for homology B, and rare restriction sites such as MluI, PmeI and Pac I for mapping of the modified BAC.
- pBV.IRES.LacZ.PA This vector is designed to introduce lacZ marker gene into a coding exon or the 3' UTR of a given gene, to study gene expression and gene regulation in vivo. IRES will enable the translation of the marker gene independent of the endogenous translation initiation codon.
- pBV.EGFP1 (FIG. 10) This vector is designed to introduce the brighter version of the green fluorescent protein, EGFP1 (Clontech), into an exon of a given gene before the endogenous ATG or fused in frame with the endogenous gene.
- the green fluorescent protein will mark gene expression in living cells and living organisms. Since the marker gene does not contain its own polyA addition sequence, the endogenous polyA sequence is used.
- pBV.IRES.EGFP1 (FIG. 11) This vector is used to introduce EGFP1 gene into the coding region or the 3' UTR of a given gene, with its translation independent of the endogenous translation frame.
- pBV.pGK.Neo.PA This vector is designed to introduce a neo expression cassette into the BAC, containing the neo gene with the pgk promoter and the polyA addition signal.
- Modified BAC can be introduced into tissue culture cell lines (i.e. ES cells) to obtain stable transfected cells by selecting for neomycin resistance.
- This vector is particularly useful for gene targeting with modified BACs. Notice that although there are two identical pgkpA sequence at the 3' end of the neo gene, it will not interfere with the proper expression of the neo gene. The only consequence is that during BAC modification, one of the pgkPA sequence may be deleted due to homologous recombination.
- This plasmid vector was modified from the pMBO96 vector originally constructed by O'Connor et al (Science, 1989, Vol 244, pp. 1307-1312).
- the pMBO96 vector was a gift from Dr. Michael O'Connor.
- the original vector carries tetracycline resistance, and contains a pSC101 temperature sensitive origin of replication, which allows the plasmid to replicate at 30° C. but it will cease replication and is lost at 43° C.
- the E. coli RecA gene was amplified by PCR and sub-cloned into the Bam HI site, to create the pSV1.RecA vector.
- the Sal I site is used to subclone the recombination cassette from the building vector.
- the first step of targeted BAC modification involves the subcloning of two small genomic fragments (A and B) into an appropriate building vector, which includes two steps of conventional sub-cloning.
- a and B small genomic fragments
- PCR amplified fragment with appropriate restriction sites designed at the end of the PCR primer is the method of choice. Frequently, an additional restriction site is designed into one of the two PCR primers to assist in determining the orientation of the cloned PCR fragment. The relative imprecision of PCR amplification does not appear to affect the BAC modification efficiency.
- the following plates Prior to cloning the recombination cassette into the shuttle vector, the following plates are usually prepared: the tetracycline (10 ug/ml) LB agar plates and the tetracycline (10 ug/ml)+chloramphenicol (12.5 ug/ml) LB agar plates. Plates are made according to standard protocol [Sambrook et al., (1989) supra].
- pSV1.RecA and building vector midi-prep DNA by the alkaline lysis method (see above).
- pSV1.RecA vector Qiagen columns can also be used to obtain high purity DNA, though yield is usually low. This is due to the low copy number of the pSV1 plasmid.
- the culture should be grown at 30° C. in LB+tetracycline (10 ug/ml).
- the final midi-prep DNA is usually dissolved in 200 ul TE or ddH 2 O.
- the reaction is performed at 37° C. for >6 hours (usually overnight), then 30 units more Sal I is added, and the digestion continue for another 1-2 hours. (Optional) A small sample of the digestion (5 ul) may be run on a gel to ascertain that a complete digestion has been achieved.
- Sal I is inactivated by heating to 65° C. for 15 minutes.
- the vector is then treated with alkaline phosphatase by adding 20 ul 10 ⁇ dephosporylaiton buffer, 4 ul (1 unit/ul) calf intestinal alkaline phosphatase (Boehringer Mannheim) for 30 minutes at 37° C.
- the enzyme is then inactivated by adding 20 ul 50 mM EDTA (to a final concentration of 5 mM), and heating at 75° C. for 15 minutes.
- the digested pSV1 vector and pBV with recombination cassette are run on a 1% low melting Seaplaque GTG agarose at 75 V for 8-10 hours.
- the DNA should be run in a large well created by taping together several teeth of the comb.
- Ligation reaction Each ligation reaction is done in 20 ul total volume containing: >50 ng pSV1.vector, 100-200 ng insert, 2 ul 10 ⁇ ligation buffer (Boehringer-Mannheim), 2 ul 10 mM ATP, 1 ul ligase (Boehringer-Mannheim) and ddH2O. Ligation is carried out at 16° C. overnight.
- Transformation of DH5a competent cells with pSV1 vectors Half of the ligation reaction (10 ul) is used for transformation, by adding to 100 ul of cold, chemical-induced DH5a competent cells. Incubate 15 minutes on ice, then heat shock at 37° C. for 2 minutes, add 1 ml LB to the tube, and shake at 30° C. for 30 minutes. The cells are then centrifugated at 6000 ⁇ g for 4 minutes and the pellet is resuspended in 100 ul LB and spread onto Tet (10 ug/ml) LB agar plates. Incubate the plates at 30° C. for >15 hrs hours.
- Bacterial incubator set either at 30° C. or at 43° C.
- Shakers set either at 30° C. or at 43° C.
- the following reagents and plates should be prepared prior to the targeted modification experiment. All the plates can be stored in 4° C. for up to one month. Detailed methods for preparation of various antibiotic resistant plates can be found in Maniatis.
- Tetracycline stock solution 1000 ⁇ : 10 mg/ml in 50% ethanol, wrapped in aluminum foil and stored in -20° C. for up to one month.
- Chloramphenicol stock solution 1000 ⁇ : 12.5 mg/ml, dissolved in ethanol (>50%), stored in -20° C.
- Tetracycline plates (tet plates): LB agar plates containing 10 ug/mil tetracycline. Store in 4° C. and wrapped in aluminum foil to avoid the light.
- Chloramphenicol plates (Chl plates): LB plates contain 12.5 ug/ml Chloramphenicol.
- Tetracyline+Chloramphenicol plates LB plates contain 10 ug/mil tetracycline and 12.5 ug/ml chloramphenicol.
- a chemical method is used to prepare competent cells from BAC containing bacteria host (Inoue et al, Gene 96, p23-28, 1990).
- TB media (10 mM Pipes, 55 mM MnCl 2 , 15 mM CaCl 2 and 250 mM KCl), all the components except for MnCl 2 are mixed and the pH is adjusted to 6.7 with KOH. Then, MnCl 2 was dissolved, the solution was sterilized by filtration through a 0.45 u filter unit and stored at 4° C. All salts were added as solids.
- the cell pellet was gently resuspend in 4 ml of TB supplemented with 7% DMSO. Incubate on ice for 10 min, then dispense 0.5 ml aliquot and immediately frozen by immersion into liquid nitrogen. The tubes are stored in -80° C. for further use.
- a thick lawn of bacteria will grow on the plates incubated in 30° C.
- Pick 20 of these large colonies inoculate each colony to 2 ml LB supplemented with tet (10 ug/ml) and chloramphenicol (12.5 ug/ml), and streak the same colony onto a tet+chl plates.
- Midi-prep DNA are prepared for the positive clones by the alkaline lysis method as described above. Restriction digests and Southern blots are performed to confirm targeting event on both homology side (A and B).
- Pulse field gel analyses should be done to confirm the modification event and to determine if there are any rearrangements in the modified BACs. Since there are two Not I site flanking the BAC insert (Research Genetics), digestion with Not I should reveal the size of the modified BAC. Generally MluI, PacI and PmeI sites are included in the recombination cassette. Digestion with these enzymes will confirm the targeting events. Double digestion with these enzymes and with Not I will help to determine the integration site of the recombination cassette in the BAC. XhoI is usually used to fingerprint the modified BAC, since it has a wide distribution of fragment sizes. Comparing the Xho digestion pattern of the modified BAC with the original BAC will reveal any gross rearrangements in the modified BAC.
- Probes used to hybridized to the PFGE blots include: insert specific probes (s.a. lacZ, PolyA, GFP and Neo) and whole BAC probe (to reveal all the digested bands from the BAC). Once the modified BACs are confirmed to have the specific targeted modification events and the lack of rearrangements, these BACs are ready to be used for the biological experiments, such as producing transgenic mice or transfecting cells.
- Digestion is carried out at 37° C. for >10 hrs.
- Purified DNA is stored at 4° C. It is stable for weeks (e.g., no degradation was detected after 3 weeks).
- BACs are useful as tools for studying the regulation of gene expression in vivo.
- a BAC can include the murine brain specific zinc finger gene, RU49 [Yang et al., Development 122:555 (1996)].
- RU49 has been shown by in situ hybridization to be expressed in the granule cell population of the murine cerebellum, the dentate gyrus and the olfactory bulb in the brain.
- proper expression of the lacZ marker gene could not be obtained in the cerebellum with a 10 kb RU 49 promoter-lacZ construct in transgenic mice, e.g., only one out of ten lines showed partial expression in the cerebellum.
- the E. coli recA gene was introduced into the temperature sensitive shuttle vector.
- the host strain becomes conditionally competent to perform homologous recombination allowing in vivo modification of the resident BAC.
- FIG. 1 illustrates the steps involved in inserting a marker gene, e.g., IRES-lacZ-pGKpolyA (ILPA), into the BAC.
- a marker gene e.g., IRES-lacZ-pGKpolyA (ILPA)
- ILPA IRES-lacZ-pGKpolyA
- This shuttle vector is then transformed into E. coli containing the BAC.
- the transformants can be selected by tetracycline resistance (carried by pSV1.RecA) and chloramphenicol resistance (carried by the BACs) at 30° C. Since the shuttle vector also carries the recA gene, homologous recombination can occur between the shuttle vector and the BAC, through either homology at A or B to form co-integrates. The co-integrates are selected by growth on tetracycline and chloramphenicol plates at 43° C.
- This temperature is non-permissive for shuttle vector replication, so that the non-integrated, free shuttle vectors are lost, resulting in the selection for bacteria carrying the integrated shuttle vectors, (either into the BACs or into the bacterial chromosomes). Correct BAC co-integrates can be identified by Southern blot analyses.
- the co-integrates are then restreaked onto the chloramphenicol plates and grown at 43° C. overnight. A fraction of the co-integrates will undergo a second recombination event (resolution), through either homology at A or B.
- the resolved BACs will automatically lose the tet and the recA genes, since the excised shuttle vector plasmids cannot replicate at the non-permissive temperature.
- the resolved BACs can be selected by growing on chloramphenicol and fusaric acid plates at 37° C., as growth on fusaric acid plates selects for the loss of tetracycline resistance, i.e., counterselecting against BACs that are resistant to tetracycline. As illustrated in FIG.
- the resolved BAC can be either the original BAC or the precisely modified BAC.
- the desired clones can be identified by colony hybridization using a labeled probe for the inserted marker.
- the recA gene is only temporally introduced into the bacterial host. Once the modification is finished, the bacteria will automatically lose the recA gene, returning to the recombination deficient state suitable for stable maintenance of the modified BACs.
- This strategy termed targeted modification of BACs was tested by introducing the IRES-lacZ-polyA (ILPA) marker into the 131 kb murine BAC169 containing the RU49 locus (FIG. 2A).
- the marker gene to the first coding exon of the RU49 gene was targeted with homology fragments being 1 kb and 1.6 kb respectively (FIG. 2B).
- Placing the IRES sequence before the lacZ gene ensures the translation of the marker gene even when lacZ gene is placed after the translation start site [Pelletier et al., Nature 334:320 (1988)].
- the pSV1.RecA temperature sensitive shuttle vector containing the recombination cassette was transformed into the DH10 E.
- the co-integrates are then resolved as described above by growing the cells first on chloramphenicol plates at 43° C. and then on chloramphenicol and fusaric acid plates at 37° C. Fusaric acid provides a strong counterselection against bacteria containing the tetracycline resistance gene. Indeed, 200 colonies picked from these plates were all tet sensitive, indicating the stringency of the selection. Duplicated colonies growing on the chloramphenicol plates were used for colony hybridization with the pgkpolyA probe. Eight out of 200 colonies were positive (4%). Southern blot analyses using either homology at A1 or B1 as the probe showed that all these clones contained correctly resolved BACs (FIGS. 3C and 3D). Three BACs (lanes 4, 5 and 8) also contained wild type bands, which may represent either contamination from other clones, or a BAC containing two copies of co-integrates that resolved through two different homologous regions.
- FIG. 4 shows pulsed field gel mapping of the modified BAC L1 and L2 and the original BAC 169.
- the same filter was probed separately with the whole BAC169 probe, with a probe from the inserted marker gene (pgkpolyA) and a probe from the 5' non-modified region of the RU49 gene (A2).
- BAC169 probe (left panel) hybridizes with all the restriction fragments for each BAC.
- XhoI digestion reveals a finger print of the modified BACs showing that essentially all fragments are preserved.
- the fragment containing the ILPA insert is slightly smaller than the corresponding wild type fragment due to the replacement of the 7 kb RU49 fragment with the 4 kb marker gene (FIG. 2B).
- Digestion with NotI which releases the entire BAC insert, also reveals a slightly smaller DNA insert in modified BACs for the same reason. Since the marker gene was engineered to carry an additional PmeI site (FIG. 2), digestion of the BAC L1 and L2 DNAs with this enzyme results in the generation of two fragments, in contrast to the single fragment seen in the original BAC69.
- the sizes of these fragments allow the determination that these BACs contain approximately 75 kb 5' to the PmeI site, and 53 kb 3' to it (FIG. 2). No apparent rearrangements have occurred during the modification procedure.
- both modified BACs and BAC169 were probed with both a marker specific probe (pgkpolyA) and a probe near the promoter region and outside the modification region (A2). Consistently, both modified BACs contained a single band homologous to the marker gene probe which is not present in BAC169. When the A2 probe was used, a single band of expected size appeared in all three BACs. Additional fingerprinting of all eight modified BACs with HindIII, EcoRI and AvrII digests showed that no detectable rearrangements or deletion existed in these BACs. Thus, the temporary introduction of the recA gene into the BAC host strain does not introduce any rearrangements or deletions.
- the BAC L1 was further modified by replacing the IRES-lacZ sequence with pgk-neo sequence. In this case, homologous fragments of about 500 bp each were used.
- the modified BACs were also efficiently obtained and shown not to have any rearrangements or deletions. Therefore, targeted BAC modification is a simple method to precisely modify BACs without introducing any unwanted changes in the BACs.
- transgenic mice carrying the modified BAC169 with the IRES-LacZ insertion were generated.
- an appropriate injection buffer e.g., 100 mM NaCl, 10 mM Tris.HCl, pH 7.5 and 0.1 mM EDTA (FIG. 5A).
- the purified fractions using the SEPHAROSE CL-4B column contained a large quantity of high concentration linear DNA (e.g., 0.5 mls of 3 ⁇ g/ml DNA or more).
- the purified DNA could be directly visualized with ultraviolet light after ethidium bromide staining.
- the SEPHAROSE CL-4B column could also efficiently separate the degraded DNA (in this case in fractions 3-6) from the pure linear DNA (fractions 7-9) (FIG. 5A).
- Fraction 8 contained 3 ⁇ g/ml DNA and was used directly for pronuclear injection.
- Pronuclear injection into the fertilized C57BL/6 mouse zygote is performed according to a standard protocol [Hogan et al., in Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, New York, 1986)]. Two different concentrations of fraction 8 BAC DNA (obtained as described above) were used: 3 ⁇ g/ml and 0.6 ⁇ g/ml. No newborns were obtained with the high concentration DNA, suggesting that the high concentrations may be toxic to the zygote. However, with the lower concentration of pure linear DNA, 15 newborn mice were obtained and two of them (13%), Y7 and Y9, contained the lacZ marker gene as demonstrated on a Southern blot (FIG. 5B). The intensity of the bands allows an estimate of 2-3 transgene copies for Y7 and one copy for Y9.
- both ends of the BAC ends were assayed for in the transgenic mice. Since both BAC ends contain some vector sequence, PCR primers specific to the vector sequence were generated and used to amplify the transgenic DNA. The amplified products were then probed with a third labeled oligonucleotide probe within the amplified region. As shown in FIG. 5C and FIG. 5D: Y3, Y7 and Y9 have both ends present, while the negative controls do not. Since Y7 and Y9 also have the lacZ gene, they are likely to contain intact BAC transgenes. For Y3, whereas it has both ends it does not contain the lacZ gene. This may be due to either a rearrangement or fragmentation during the injection prior to integration.
- the Y7 transgenic mice also gave rise to germline transmission after breeding with B6/CBA mice. In two litters having a total of eight pups, three pups carried the LacZ transgene (FIG. 5E). Further analysis demonstrated that the transgene was transmitted in a Mendelian distribution to more than fifty Y7 offspring.
- lacZ gene in the cerebellum of the Y7 transgenic mice was determined by whole mount lacZ staining.
- RU49 is normally expressed in the granule cells of the cerebellum, the dentate gyrus and the olfactory bulb (including the subventricular zone, the rostral migratory stream, and the olfactory bulb proper) [Yang et al., Development, 122:555-566 (1996)].
- RU49 promoter lacZ transgenic mice with 10 kb promoter had been generated. However, all of the transgenic lines showed strong positional effects: either they did not express in the brain at all, or they were ectopically expressed in the cortex, but not the cerebellum.
- the lacZ marker gene is also expressed in the dentate gyrus and the rostral migratory stream and the olfactory bulb (FIGS. 6C and 6D).
- the pattern of the BAC transgene expression closely resembles the endogenous RU49 expression pattern in the brain. It is evident that the large genomic DNA in the BAC transgene can overcome the positional effects and confer the proper expression of RU49 in vivo, in contrast to our results using conventional transgenic constructs.
- bacterial based artificial chromosomes are ideal for constructing large DNA for gene targeting.
- BACs and PACs can be readily modified to introduce selection genes, marker genes, and deletions.
- Making a BBPAC gene targeting construct will take about the same time as making a conventional targeting construct (1-3 months).
- BBPAC targeting construct DNA can be easily isolated in milligram quantity and high quality. This is advantageous over the YAC system, since it is difficult to purify large quantities of high quality YAC DNA.
Abstract
Description
______________________________________ 500 ml TB 1 L TB ______________________________________ Tap H.sub.2 O 500 ml 1 L (not distilled H.sub.2 O) Bacto tryptone 5 g 10 g Yeast extract 0.5 g 1 g Glucose 0.5 g 1 g NaCl 4 g 8 g 0.1 M ZnCl.sub.2 0.25 ml 0.5ml Chlorotetracycline 4ml 8 ml (6.3 mg/ml) Bacto agar 7.5 g 15 g ______________________________________
______________________________________ 500 ml TB 1 L TB ______________________________________ NaH.sub.2 PO.sub.4.H.sub.2 O (1 M) 36 ml 72 ml Fusaric Acid (2 mg/ml, 3ml 6 ml filter ster.) Chloramphenicol 0.5ml 1 ml (12.5 mg/ml) ______________________________________
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US08/880,966 US6143566A (en) | 1997-06-23 | 1997-06-23 | Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof |
US09/007,206 US6130090A (en) | 1997-06-23 | 1998-01-14 | Methods of performing gene trapping in bacterial and bacteriophage-derived artificial chromosomes and use thereof |
US09/102,488 US6156574A (en) | 1997-06-23 | 1998-06-22 | Methods of performing gene trapping in bacterial and bacteriophage-derived artificial chromosomes and use thereof |
AU79848/98A AU730859B2 (en) | 1997-06-23 | 1998-06-23 | Methods of preforming homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof |
JP50494899A JP2002515764A (en) | 1997-06-23 | 1998-06-23 | Method for preforming modifications based on homologous recombination of nucleic acids in non-recombinant cells and use of the modified nucleic acid products |
EP98930459A EP0998574A1 (en) | 1997-06-23 | 1998-06-23 | Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acids |
PCT/US1998/012966 WO1998059060A1 (en) | 1997-06-23 | 1998-06-23 | Methods of preforming homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof |
CA002294619A CA2294619A1 (en) | 1997-06-23 | 1998-06-23 | Methods of preforming homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof |
US09/648,933 US6485912B1 (en) | 1997-06-23 | 2000-08-24 | Methods of performing gene trapping in bacterial and bacteriophage-derived artificial chromosomes and use thereof |
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